The invention relates to [NiFe]-hydrogenases having an improved resistance to dioxygen (O2).
The use of hydrogen as a first energy vector is now widely recognized as a long-term solution from the point of view of clean and sustainable energy economy (CHORNET & CZERNIK, Nature, 418, 928-9, 2002).
Some photosynthetic organisms, belonging to the group of the green algae or cyanobacteria, possess hydrogenases, catalyzing the conversion between H+ and H2. The presence of hydrogenases confers to these organisms the ability to produce dihydrogen, starting from solar energy and using water as electron and proton donors. This type of biological conversion of light energy into hydrogen is one of the most efficient in terms of energy conservation, as 10% of the incident light energy can theoretically be recovered in hydrogen (PRINCE & KHESHGI, Crit. Rev. Microbiol., 31, 19-31, 2005). Due to their high catalytic turnover and their specificity towards H2, hydrogenases are also envisioned as potential catalysts to replace platinum in fuel cells, through the design of so-called bio-fuel cells. Such a design might considerably alleviate the price of fuel cells in which both platinum and H+ selective membranes represent the major costs. It has also been proposed to use hydrogenases in designing biosensors, for safety applications (QIAN et al., Biosens Bioelectron, 17, 789-96, 2002; BIANCO, J Biotechnol, 82, 393-409, 2002). Thus, potential applications of hydrogenases include dihydrogen photoproduction, bio-fuel cells and biosensors.
Hydrogenases constitute a family of oxidoreductase enzymes which have been classified according to the metal content of their active sites (VIGNAIS et al., FEMS Microbiol Rev, 25, 455-501, 2001). The main classes are [NiFe]-hydrogenases and [FeFe]-hydrogenases, which are phylogenetically distinct families of proteins.
[NiFe]-hydrogenases have been isolated from diverse bacteria including for instance Desulfovibrio, Azobacter, Rhodobacter, Ralstonia, Rhizobium, Bradyrhizobium, and Synechocystis. These enzymes are also found in several archaea such as Methanococcus, Methanosarcina, Acidianus, Pyrobaculum. They typically comprise a large subunit, containing the Ni—Fe active site, and a small subunit containing [Fe—S] clusters involved in the electron transfer; they may also contain additional subunits. The large subunits of all [NiFe]-hydrogenases appear to be evolutionary related. They contain at least four conserved motifs, designated (from N-terminal to C-terminal) as L1, L2, L3, and L4. In certain archaea, the large subunit is truncated before L4, which motif is then contained in an additional very small subunit. A fifth conserved motif, designated as L0, is also found near the N-terminal end of most of [NiFe]-hydrogenases (KLEIHUES et al., J. Bacteriol., 182, 2716-24, 2000; BURGDORF et al., J. Bacteriol., 184, 6280-88, 2002).
These motifs are defined by the following consensus sequences (one-letter code):                L0: R[I/V/A]EG[H/D/A]        L1: RGXE, wherein X=L, I, F, V or M        L2: [R/K]X1C[G/R]X2C, wherein X1 is any amino acid residue, X2=L, V, I or M; L1 and L2 being separated by 16 any amino acid residues;        L3: X1X2X3X4X5X6X7X8X9X10X11X12[D/S/E], wherein X1=D, S, N or E, X2=H, D, S, N or L, X5=H, S, A, Q or W, X6=F, T, Y or G, X9=L, F, M or Y, the other Xn being any amino acid residue;        L4: D[P/I/S]CX1X2CX3X4[H/R], wherein X2=A, S, V, G or T, X1, X3 and X4 are any amino acid residue.        
In said motifs, [aa1/aa2 . . . ] means that said amino acids are alternatives at a given position; any amino acid residue refers to a natural or synthetic amino acid including enantiomers and stereoisomers of any of the 20 usual amino acids; Xn corresponds to all the other positions not specifically mentioned.
Each of motifs L2 and L4 contains two cysteine residues which are involved in the binding of nickel in the active site. Although L3 is rather variable, it is easily identified in a multiple sequence alignment by ClustalW [Chema R, Sugawara H, Koike T, Lopez R, Gibson T J, Higgins D G, Thompson J D (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res, 31:3497-3500]. The sequence of L3 is DHLVHFYHLHALD in D. fructosovorans [NiFe]-hydrogenase and SHALSFFHLSSPD in the Synechocystis PCC6803 enzyme.
Both [NiFe]-hydrogenases and [FeFe]-hydrogenases are O2 sensitive. Generally, [Fe]-hydrogenascs are irreversibly inactivated by O2. In contrast, O2-exposed [NiFe]-hydrogenases can be reactivated, and there are some examples of relatively oxygen-tolerant enzymes, such as MBH and SH from the Knallgas bacterium Ralstonia eutropha although they tend to be less active (BURGDORF et al., J Mol Microbiol Biotechnol, 10, 181-96, 2005). A sub-group of highly oxygen-tolerant but even less active [NiFe]-hydrogenases are the H2-sensors such as RH from Ralstonia eutropha (BERNHARD et al., J Biol Chem, 276, 15592-7, 2001) and the HupUV proteins from Rhodobacter capsulatus (ELSEN et al., J Bacteriol, 178, 5174-81, 1996) and Bradyrhizobiurn japonicum (BLACK et al., J Bacteriol, 176, 7102-6, 1994), as well as [NiFeSc]-hydrogenases.
The sensitivity of hydrogenases to O2 represents a major obstacle to the development of technological applications of these enzymes. For instance, because of hydrogenase inhibition by the dioxygen produced during water photolysis (LEGER et al., J Am Chem Soc, 126, 12162-72, 2004), photosynthetic production of dihydrogen is only a transient phenomenon under natural conditions (COURNAC et al., J Bacteriol, 186, 1737-46, 2004). As a result, actual dihydrogen production efficiencies obtained in laboratory experiments are lower than 1% (MELIS et al., Plant Physiol, 122, 127-36, 2000; FOUCHARD et al., Appl Environ Microbiol, 71, 6199-205, 2005).
Both [FeFe] hydrogenases and [NiFe]-hydrogenases possess hydrophobic gas channels allowing H2 and also O2 diffusion between the molecular surface and the active site. FIG. 1 represents the crystallographic structure of the prototypic [NiFe] hydrogenase from D. fructosovorans: the gas channels are shown in grey.
At the internal end of the hydrophobic channels, near the [NiFe] active site, two hydrophobic residues, usually a valine and a leucine that are conserved in oxygen-sensitive [NiFe]-hydrogenases, are respectively replaced by isoleucine and phenylalanine in the sub-class of oxygen-tolerant H2-sensors (VOLBEDA et al., Int. J. Hydrogen Energy, 27, 1449-61, 2002). These correspond respectively to residue X2 of the conserved motif L2, and X9 of the conserved motif L3. It has been hypothesized that the presence of bulkier residues in the oxygen-tolerant hydrogenases may reduce the channel diameter at this point, thus limiting access of O2 molecules, which are larger than H2, to the active site. This hypothesis is now confirmed by independent studies on two different RHs (H2 sensors) by Buhrke et al. (J. Biol. Chem. 2005, 280, 23791-23796) and Duché et al; (FEBS J. 2005, 272, 3899-3908)
On this basis, it has been suggested to modify naturally occurring hydrogenases in order to improve their resistance to dioxygen, by reducing the diameter of their H2 channels. PCT application WO 2004/093524 thus proposes to modify [FeFe] hydrogenases by substituting the residues lining the H2 channel by bulkier residues, such as tryptophan or phenylalanine.